The growth of axons and the formation of synaptic connections are associated with the expression of the neuronal membrane phosphoprotein, GAP-43. Yet while the relationship of this protein to growth and many of its biochemical properties are well-established, very little is known about how its expression is controlled in the neuron. Our preliminary data suggest that GAP-43 expression is regulated primarily by post-transcriptional mechanisms, that operate on the rate of degradation of the mRNA. Here, we propose to study the nature of the physiological signals and of the molecular mechanisms that regulate GAP43 mRNA levels during neuronal differentiation. Our specific aims are: First, to investigate the nature of the extracellular signals that control GAP-43 mRNA levels during neuronal differentiation. To this end, we will determine the levels of this mRNA upon treatment of neuronal cell cultures and PCI2 cells with a variety of agents that either promote or inhibit neurite outgrowth. Second, to test the hypothesis of post-transcriptional regulation under several experimental conditions. These studies will examine the contribution of the rates of synthesis and degradation of GAP-43 mRNA on the levels of this mRNA by Northern blot analysis, run-on transcription assays, and pulse-chase labeling to determine the half-life of the mRNA . Finally, to study the molecular mechanisms that control the degradation of GAP-43 mRNA. Towards this goal, we will examine the specific RNA sequences and cytoplasmic factors that contribute to the NGF-mediated stabilization of GAP-43 mRNA in PCI2 cells. These studies will be performed using transfection experiments with constructs containing various amounts of coding and non-coding sequences of GAP-43 mRNA, and RNA gel retardation assays. The studies proposed here will provide fundamental information on the extracellular signals and intracellular mechanisms that control the degradation of GAP-43 mRNA during neurite outgrowth. Given that most of the research on the control of expression of neural genes has been focused almost exclusively on the transcriptional aspects of the regulation, our studies on the post-transcriptional regulation of GAP-43 will provide a first and unique opportunity to examine an area of research that has been so far poorly explored. The identification of specific sequences and factors that control the degradation of GAP-43 mRNA, will clearly contribute to establish the role of post-transcriptional mechanisms in controlling gene expression during neuronal differentiation.